The production of thin sections of cryopreserved specimens, in particular biological specimens with vitrified material (i.e. solidified in glassy fashion), is important for cryoelectron microscopy and similar investigation methods. Cryopreserved specimens largely avoid distortions that can derive from desiccation, chemical modifications (including contrast agents), and other specimen stabilization methods and therefore enable investigation of the specimen at the ultrastructure scale in a state that comes very close to the living state of the initial specimen. A very thin section thickness is required for investigation using an electron microscope or the like, however, namely tens to a few hundred nanometers depending on the type of specimen. This, along with the deep-frozen state (generally below −100° C.) of the sections, places serious demands on the user's skill and on the quality and precision of the sectioning device.
In addition to the actual sectioning operation, removing the sections from the edge of the knife and applying them onto a specimen carrier (referred to here as a specimen support) constitutes a particularly delicate operation. The specimen supports used for an electron microscope are generally grids made of a thin metal foil. For room-temperature applications the possibility exists, for example, of carrying the sections away from the edge on the surface of a water bath in which the sectioning apparatus is partly immersed; this offers the additional advantage that sections crimped during the sectioning operation can relax again (decompress) on the water surface, and are only then received by the grid. The water-bath approach is not available for low-temperature applications, and transport from the knife edge to the grid must therefore be effected in some other manner.
Because manipulation of sections in a cooled preparation chamber of an ultramicrotome is performed manually, especially during the sectioning operation, such manipulation requires an experienced user. Because the sections are produced in a sequence of many sectioning motions rather than individually, the sections are produced in the form of an interconnected strip of a plurality of individual sections. An interconnected strip of this kind is then pulled with a suitable tool, for example a hair mounted at the end of a wooden rod, from the knife edge over a grid and then immediately laid down. Accurate positioning of the grid with respect to the knife blade is necessary so that the fragile section strip can be picked up close to the knife edge.
A known device for producing low-temperature thin sections is the Leica EM FC6, available from the assignee of the present application, Leica Microsystems, GmbH, of Hernalser Hauptstrasse 219, A-1170 Vienna, Austria. The sectioning operation takes place in a working space with at least one side wall that is adjacent or thermally coupled to a cooling chamber Tillable with a coolant (e.g. liquid nitrogen), or where the working space is surrounded on several sides (e.g. in cupped fashion) by the cooling chamber. The working space cooled in this fashion is open toward the top and thus accessible to the user. The specimen to be sectioned is mounted on a specimen holder positioned on one vertical side of the working space, to which holder a vertical motion can be imparted in the manner of a vibrating head. The sectioning knife is unmoved during the sectioning operation and is positioned by means of a knife holder mounted in the floor of the working space (shifting for alignment purposes is possible). The knife holder furthermore contains instruments for retaining a grid (or a few grids), in particular a grid holder into which the grid can be clamped at its edge. This grid holder is shiftable in a horizontal direction toward and away from the knife; positioning consists solely in sliding toward the knife. An end stop prevents contact between the grid and the knife. The specimen preparation device located in the working space can furthermore comprise an apparatus having a preparation surface, in which grids are set in place and pressed while still in the working space. Placement of the grid onto a preparation surface of this kind, in particular the operation of pressing the sections onto the grid (specimen support), can, however, generate contaminants.
In order to counteract electrostatic charging of the sections in the cooled working space, the Leica EM FC6 provides an ionizer that slightly ionizes the gases (vapors) surrounding the material to be sectioned, in order to ensure dissipation of electrical charges.
With other, earlier models, holders for multiple specimen supports were provided. These holders had a coarse vertical displacement capability, and (by shifting) a coarse displacement capability in the horizontal plane. These models, as well, comprised a section press integrated into the specimen preparation device.
The aforesaid known devices have the following disadvantages:
Guidance of the section strip and positioning of the grid are performed manually, and are thus susceptible to operating errors, and require considerable user skill.
The numerous manipulation steps on the sections within the working space are laborious and can result in section losses and contamination.
The grid is retained on the side facing the knife edge. This impedes placement of the section strip, since the section strip is pulled from the knife edge toward the holder.
The effect of the ionizer is impaired by components made of solid metal for retaining the grid.
There is a risk of contaminating the sections with condensed ice or other contaminants from the environment as a result of manipulation that lasts too long.
The article “Vitreous cryo-sectioning of cells facilitated by a micromanipulator,” by M. Ladinsky et al., J. Microsc. 224 (2006) 129-134 describes a cryomicrotome arrangement having a micromanipulator. In it, a fiber retained by the micromanipulator at the end of a wooden rod serves to pick up the sections and guide the section strip to the support. The manipulator is mounted on the equipment table, not on the cooling chamber itself. Because a vibration damping system is present between the equipment table and cooling chamber, relative motions occur between the manipulator and the ultramicrotome with cooling chamber; in particular, contact by the user (e.g. operation of the microtome arrangement's stereomicroscope) inevitably causes such relative motions.
DE 202 21 696 U1 describes a microscope arrangement having a micromanipulator for carrying out microscopic manipulations and injections on living material. The micromanipulator is attached to the microscope by means of an adapter. DE 1279 368 A describes a micromanipulator for moving and producing small tools under a microscope, the micromanipulator being arranged, by means of a magnetic tool holder, at the lower objective end of the microscope.